Proteasomes (high -molecular-weight protease) were purified from rat skin, and their enzymologic properties, gross structure, and tissue distribution were investigated. Skin proteasomes were purified by successive (NH4)2S04 fractionation and by phenyl Sepharose CL-4B and HPLC gel filtration chromatography. The molecular weights of the proteasomes were estimated from gel filtration to be 750 kD. On sodium dodecylsu lfate -~olya~ryla~ide gel electrophoresis, th~ ~u rified enzymes dissoCiated mto

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... everal bands, the maJonty falling into the range of 36 -20 kD . Two-dimensional electrophoretric analysis demonstrated approximately 10-15 separate protein spots with pi values varying between 3 and 10. As analyzed by electron microscopy, the gross structure of the enzymes showed an almost symmetrical ring-shaped particle with a small hole in the center. Succynyl-leucyl-Ieucylvalyl-tyrosine-4-methylcoumaryl-7 -amide, a fluorogenic P roteasomes are nonlysosomal enzymes with high molecular weights (Mr = 750 kD). They have been found as multicatalytic proteinase complexes [1) and have been shown to play an essential role in an energy-dependent non lysosomal proteolytic pathway responsible for the selective removal of unnecessary proteins generated in cell s [2 -6). Proteasomes are w idely distributed in various eukaryotic cells and tissues ranging from humans to yeast [7, 8) . Moreover, recent studies have reported their localization in the nucleus and cytoplasm [9,10). This wide intracellular distribution may be of significance, because the physiologic functions of proteasomes are still unknown. Previous ly, Tanaka et al (7) showed the existence of proteasomes in rat skin by quantitative enzyme immunoassay. In this study, we report the purification of rat skill proteasomes and describe some of their properties and their gross structure from electron microscopy, as wel l as their tissue distribution as measured by enzymologic and immunologic tec hniques. MATERIALS AND METHODS Materials Ten male Wistar rats (7 weeks old , 200-250 g) were obtained from Takasugi In stitutc (Tokyo). Phenyl Sepharose C L-4B and molccular weight marker proteins were purchased from Pharmacia Co. High-performance liquid chromatography (HPLC) was carried out using a TSKG3000SWXL or TSK4000SW gel filtration column (Toyo Soda, Abbreviations: AMC, 7-amino-4-mcthylcoumarin; SLLVY-MCA, succyn y l-leucy l-l cucyl-val y I-tyrosine-4-methylcoumary 1-7 -amide. substrate for serine proteinases, demonstrated the highest activity in terms of substrate specificity. Sodium dodecylsulfate, Ca++, and some free fatty acids activated enzyme activity. Activity was inhibited by diisopropylfluorophosphate, leupeptin, N-ethylmaleimide, iodoacetamide, and chymostatin. These results show that both serine and cysteine residues are related to the enzyme activity of proteasomes. Total and specific enzyme activities in the epidermis were, respectively, 10 and 20 times higher than in the dermis. Immunohistochemical studies utilizing the avidin-biotin complex method with monoclonal antibody revealed that the enzyme is distributed throughout the epidermis. These findings indicate the epidermal localization of proteasomes. Key words: nonlysosomal enzyme/rat skin/multicatalytic proteinase complex.] Invest DermatoI101 : 346-351 , 1993